The overall goals are to elucidate the molecular mechanisms by which cholera toxin activates adenylate cyclase in intact eukaryotic cells and to generate V. cholera mutants that produce defective "toxins". More specifically, we are interested in the mechanism by which subunit A of cholera toxin activates adenylate cyclase, the mechanism for penetration of subunit A (or intact toxin) to the inner surface of the plasma membrane or to the cytosol and the precise structural features of the subunits of cholera toxin that are critical for adenylate cyclase activation and cell penetration. In these studies we will employ affinity chromatography and other techniques to identify, purify and characterize proteins in the cell membrane or cytosol that selectively bind subunit A. We will use toxin mutants to correlate structure and function and we will determine if appropriate V. cholera mutants will protect against the natural disease as this is manifested in experimental animals.